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Endocrine, Metabolic & Immune Disorders -Drug Targets
ISSN: 1871-5303
eISSN: 2212-3873
Impact
Factor:
1.897
Faruk Kutluturk1,*, Ahmet Inanir2, Aydın Rustemoglu3, Suheyla U. Kaya4, Ayse K. Demir4,
Gul Dursun3 and Serbulent Yigit3
1Department of Endocrinology and Metabolism, Gaziosmanpasa University School of Medicine, Tokat, Turkey;
2Department of Physical Therapy and Rehabilitation, Gaziosmanpasa University School of Medicine, Tokat, Turkey;
3Department of Medical Biology, Gaziosmanpasa University School of Medicine, Tokat; 4Department of Internal Medi-
cine, Gaziosmanpasa University School of Medicine, Tokat, Turkey
A R T I C L E H I S T O R Y
Received: September 23, 2017
Revised: December 10, 2017
Accepted: January 31, 2018
DOI:
10.2174/1871530318666180212100119
Abstract: Introductıon: Osteoporosis is a common disease, and several factors contribute to its devel-
opment. Recently, there has been increasing evidence that vitamin K (VK) play s a critical role in m ain-
taining bone strength. Vitamin K serves as a co-factor for the -carboxylation of particular proteins to
convert specific glutamic acid residues to -carboxyglutamic acid residues. This process involves two
enzymes, -glutamyl carboxylase and vitamin K epoxide reductase (VKORC1). The number of studies
investigating the effects of VKORC1 gene mutations on bone mineral density in postmenopausal os-
teoporosis patients is limited. The aim of this study was to investigate the relationship between the
VKORC1 -1639G>A polymorphism and osteoporosis in postmenopausal Turkish women.
Methods: The study group consisted of 176 postmenopausal women with osteoporosis and 140 healthy
postmenopausal women. The selection criteria for the healthy controls included non-osteoporotic bone
mineral density (BMD) and similar demographic characteristics to the osteoporosis group. The geno-
typing of the VKORC1 -1639G>A polymorphism was conducted using the restriction fragment-length
polymorphism method.
Results: We found that the genotype frequencies of the GG, GA and AA genotypes were 25.6, 64.2
and 10.2% in the patients and 33.6, 55.8 and 10.7% in the controls, respectively. In the patient and
control groups, the genotype distribution of the studied locus was found to be non-compatible with
Hardy–Weinberg equilibrium. We found a nonsignificant association between the -1639G>A poly-
morphism in the VKORC1 gene and osteoporosis in postmenopausal Turkish women.
Conclusion: We have shown that the VKORC1 -1639G>A polymorphism is not a risk factor for post-
menopausal osteoporosis.
Keywords: Vitamin K epoxide reductase, VKORC1, osteoporosis, gene, polymorphism, postmenopausal women.
1. INTRODUCTION
Osteoporosis is a common disease that is characterized
by low bone mineral density (BMD), microarchitectural de-
terioration, and increased risk of fractures [1-3]. Osteopenia
and osteoporosis are related conditions. The difference be-
tween osteopenia and osteoporosis is that in osteopenia the
bone loss is not as severe as in osteoporosis. Fractures
caused by osteoporosis contribute to morbidity, reduce the
quality of life, and lead to disability, interfering with activi-
ties of daily living [4-6]. Several factors contribute to the
development of osteoporosis, including clinical, medical,
behavioural, nutritional and genetic variables [5]. The
*Address correspondence to this author at the Department of Endocrinology
and Metabolism, Gaziosmanpasa University School of Medicine, 60200
Tokat, Turkey; Tel: +90 (507) 247 73 98; Fax: +90 (356) 212 21 42; E-mail:
fkutluturk@yahoo.com
genetic control of osteoporosis is polygenic, and previous
studies of the association of various genetic polymorphisms
(such as ACE gene I/D polymorphism, vitamin D receptor,
collagen type I alpha 1 gene, and estrogen receptor gene al-
pha) with BMD produced controversial and inconclusive re-
sults [6-12]. Substantial efforts have been dedicated to under-
standing whether variants in genes known to influence bone
physiology also influence risk for low BMD and possibly frac-
tures-called candidate genes. The candidate gene approach has
relied on the assumption that if a gene is important for bio-
logical reasons, it may also affect the trait of clinical interest.
Whether these genes do indeed influence propensity to osteo-
porosis and fracture has remained uncertain because many
candidate gene studies lacked sufficient sample sizes and a
replication group with which to validate findings [8].
Recently, there has been increasing evidence that vitamin
K (VK) plays a critical role in maintaining bone strength by
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Endocrine, Metabolic & Immune Disorders - Drug Targets, 2018,18, 281-286
281
RESEARCH ARTICLE
Association between Vitamin K Epoxide Reductase (VKORC1) -1639G>A
Polymorphism and Osteoporosis in Postmenopausal Women
282 Endocrine, Metabolic & Immune Disorders - Drug Targets, 2018, Vol. 18, No. 3 Kutluturk et al.
acting as a cofactor of the enzyme gamma carboxylase,
which in turn activates VK-dependent proteins (OC, matrix
Gla protein, Gla-rich protein, protein S, and growth arrest-
specific 6 protein) in bone [13-17].
Vitamin K serves as a co-factor for the -carboxylation of
particular proteins to convert specific glutamic acid (Glu)
residues to -carboxyglutamic acid (Gla) residues. This
process involves two enzymes, -glutamyl carboxylase and
vitamin K epoxide reductase (VKORC1) [18-20].
Vitamin K-dependent GGCX converts glutamate to a
carboxylated glutamate (Gla) of a limited number of protein
called VKDPs [21]. Whereas GGCX produces Gla residues
in the substrate protein, the co-substrate of GGCX, vitamin
K hydroquinone, is oxygenated into vitamin K 2,3-epoxide
(vit K>O). Because the dietary intake of VK is limited, vit
K>O must be recycled to vitamin k hydroquinone before it
can be reused. This reaction is catalyzed by the VKOR activ-
ity [21, 22]. Genomes of higher vertebrates possess two
paralog genes, VKORC1 and VKORC1L1, that encode en-
zymes unique in catalyzing de-epoxidation of vitamin K 2,3-
epoxide, a product of post-translational modification of
VKDPs [21, 23]. VKDPs are known to be essential for di-
verse physiological functions including hemostasis and co-
agulation, bone development and homeostasis, vascular ho-
meostasis, remodeling and calcification, cellular growth,
survival, and signaling, metabolic homeostasis, and fertility
[21]. The VKORC and GGCX are integral membrane pro-
teins that reside in the endoplasmic reticulum and together
activate VKDPs during their secretion [24].
Recent studies have demonstrated that the orphan nuclear
receptor SXR (also known as PXR, and PAR) plays a central
role in the transcriptional regulation of xenobiotic detoxify-
ing enzymes and transporters [25-27]. SXR mRNA is ex-
pressed in osteosarcoma cell lines, and VK induced the ex-
pression of the prototypical SXR target gene CYP3A4 in
these cells. Therefore, SXR is likely to be involved in the
maintenance of bone homeostasis in addition to its known
role in hormonal homeostasis. This reveals a novel biological
function for SXR and suggests that a subset of SXR activa-
tors may function as effective therapeutic agents for the
management of osteoporosis [25].
Some evidence suggests that long-term warfarin therapy
is associated with low BMD in patients compared with pa-
tients not on warfarin therapy [28-30]. Several studies have
reported lower serum levels of VK in those with osteoporotic
fractures when compared to control subjects [31-33]. In par-
ticular, a few observational studies, mainly conducted in
Asian populations, have shown that VK intake and/or plasma
levels may be associated with a reduction of undercarboxy-
lated OC, although it did not alter BMD or bone turnover
markers. [1, 31, 34-36] Therefore, VK supplementation is
still not globally recommended to prevent postmenopausal
bone loss [13, 37]. VKORC1 is located on human chromo-
some 16p11.2 [20]. Mutations in the VKORC1 gene may
modify the gamma-carboxylation of osteocalcin and may
influence BMD.The VKORC1 gene is highly polymorphic,
and numerous single-nucleotide polymorphisms (SNPs) have
been identified [38, 39]. The VKORC1 -1639G>A polymor-
phism (rs9923231) is located in the promoter of the
VKORC1 gene [40]. The aim of this study was to investigate
the relationship between the VKORC1 -1639G>A polymor-
phism and osteoporosis in Turkish postmenopausal women.
2. MATERIALS AND METHODS
The study group consisted of 176 postmenopausal
women with osteoporosis and 140 healthy postmenopausal
women. All patients were recruited at the Department of
Physical Medicine and Rehabilitation of Gaziosmanpasa
University Tokat, Turkey. For each patient, the following
demographic and clinical variables were recorded: age,
weight, height, body mass index (BMI), menopause duration,
and smoking status. All the patients were women of post-
menopausal status, which was defined as menstruation that
had stopped completely for at least 1 year. The exclusion
criteria were premature menopause, surgical menopause,
presence of malignancy, presence of systemic disease (such
as hyperthyroidism, parathyroid disorders, Crohn’s disease
and chronic inflammatory and autoimmune rheumatic condi-
tions) and the administration of drugs (such as corticoster-
oids, ACE inhibitors or L-thyroxine) that might affect bone
metabolism. The selection criteria for the healthy controls
included non-osteoporotic BMD (T score > 2.5 SD) and
similar demographic characteristics to the osteoporosis
group, such as age, menopausal age, weight, height, body
mass index (BMI), presence of systemic disease, admission
of drug use and smoking.
2.1. BMD Measurement
By current criteria, a bone mineral density at the femoral
neck equal to or less than 2.5 standard deviations below the
mean for a young person of the same sex is diagnostic of
osteoporosis. This is reported as a T-score of –2.5 or less.
Prescribing criteria for antiresorptive treatment are based
predominantly on the T-score. Women were diagnosed with
postmenopausal osteoporosis using a DEXA machine on the
basis of T score (standard deviation from young normative
BMD mean), as described by the World Health Organization
[41]. All BMD measurements were performed at the lumbar
spine (L2-L4) and at the femoral neck by dual-energy X-ray
absorptiometry (DXA) using a Holojic QDR (Hologic, Inc.,
Waltham, MA, USA).
2.2. DNA Isolation and Genotyping
Genomic DNA was extracted from blood samples with
using aPureLink™ Genomic DNA Mini Kit (Invitrogen,
Carlsbad, CA-USA). PCR was performed in a total volume
of 25 L using 100 ng of genomic DNA with 20 pmol of
each primer, 0.2 mM of each dNTP, 1X buffer, 2 mM
MgCl2, and 1 U of Taq DNA polymerase. Cycling was per-
formed in a Techne TC-4000 Thermal Cycler (Bibby Scien-
tific Limited, Staffordshire, UK) as follows: amplification
consisted of a two-minute denaturation step at 94 °C; thirty-
five 30-second cycles at 94 °C, 30 seconds at 60 °C, and 30
seconds at 72 °C; and a seven-minute final extension at 72
°C, followed by cooling to 4 °C.
The genotyping of the VKORC1 -1639G>A polymor-
phism was conducted using a restriction fragment-length
polymorphism (RFLP) method. PCR products were digested
with the specific Hap II restriction enzyme. The digested
Vitamin K Epoxide Reductase -1639G>A Polymorphism Endocrine, Metabolic & Immune Disorders - Drug Targets, 2018, Vol. 18, No. 3 283
PCR products were resolved by electrophoresis in 3% aga-
rose gels containing 0.5 g/ml ethidium bromide. Restriction
fragments were visualized with the use of a Vilber-Lourmat
QUANTUM-ST4 Gel Quantification and Documentation
System.
2.3. Statistical Analysis
Statistical analyses were performed using the Statistical
Package for Social Sciences version 20 (SPSS Inc., Chicago,
IL, USA). Descriptive statistics (means and standard devia-
tion or absolute and relative frequencies) are given for the
demographic data. The results are given as the means ± SD.
Chi-square and Fisher's exact test were used for the statisti-
cal analysis of the data, and the Bonferroni (Pc) test was per-
formed to correct the significant p values. The chi-square
(2) test was used to evaluate the genotype distributions of
the patients and controls for Hardy–Weinberg equilibrium.
The relationships between the -1639G>A polymorphism and
the clinical and demographic features of patients were ana-
lysed using the 2 test or an analysis of variance. The 2 test
was used to compare categorical variables, and odds ratios
(ORs) and 95% confidence intervals (CIs) were calculated to
assess risk. All P-values were two-tailed, and values < 0.05
were considered to indicate significance.
3. RESULTS
The baseline clinical and demographic features of the
study patients with osteoporosis and the healthy controls are
listed in Table 1. Demographic features such as age, age at
menopause, years since menopause, BMI, family history of
osteoporosis, calcium supplementation and smoking were
similar between the groups (P>0.05). The results of the sta-
tistical analysis of the allele and genotype frequencies of the
-1639G>A polymorphism in the VKORC1 gene are given in
Table 2. We found that the genotype frequencies of the GG,
GA and AA genotypes were 25.6, 64.2 and 10.2% in the
patients and 33.6, 55.8 and 10.7%, in the controls, respec-
tively. In the patient and control groups, the genotype distri-
bution of the studied locus was found to be non-compatible
with Hardy–Weinberg equilibrium (P<0.05). The GG geno-
type was less common in the patients (25.6% vs 33.6%) than
the controls, but the difference was not significant
(P=0.135). We found a nonsignificant association between
the -1639G>A polymorphism in the VKORC1 gene and
Table 1. Demographic data of patients and control groups.
Patients (N=176) Control (n=140)
N Mean (SD) N Mean (SD)
Age, years 176 57.87 (6.54) 140 57.41 (9.20)
BMI, kg/m2176 30.96 (4.11) 140 31.34 (4.50)
Femur Neck, T score 176 -2.76 (0.60) 140 0.01 (0.87)
Femur Total, T score 176 -1.55 (0.94) 140 -0.04 (0.85)
Lombar, T score 176 -0.654 (0.90) 140 0.41 (1.05)
Menopause age, years 176 46.28 (3.82) 140 46.49 (3.54)
Menopause period, years 176 3.74 (2.53) 140 4.08 (2.34)
Diagnosis age, years 176 53.11 (4.53) - -
Disease period, years 176 5.10 (5.72) - -
N Percent N Percent
Yes 13 7.38% 9 6.43%
Family history
No 163 92.62% 131 93.57%
Yes 6 2.86% 0 0
Vertebral fracture
No 170 97.14% 140 100.00%
Yes 0 0 - -
Hip fracture
No 176 100.00% - -
Yes 14 8.00% 0 0
Fracture history
No 162 92.00% 140 100.00%
Yes 9 5.11% 8 5.71%
Smoke
No 167 94.89% 132 94.29%
BMI- Body Mass Index; SD- Standart Deviation; N-Number of subjects.
284 Endocrine, Metabolic & Immune Disorders - Drug Targets, 2018, Vol. 18, No. 3 Kutluturk et al.
postmenopausal osteoporosis in a Turkish population
(P>0.05).
4. DISCUSSION
Polymorphisms of several genes, including those encod-
ing the vitamin D receptor, type I collagen, oestrogen recep-
tor, and calcitonin receptor, have been associated with BMD
[42-45]. However, conflicting results have been obtained,
and genetic susceptibility to osteoporosis is not fully under-
stood [2, 42-46].
We investigated the relationship between the VKORC1 -
1639G>A polymorphism and postmenopausal osteoporosis.
Recently, several studies have investigated the relationship
between the VKORC1 -1639G>A polymorphism and many
diseases. It has been reported that the VKORC1 -1639G>A
(rs9923231) gene polymorphism is associated with warfarin
response [47-51], increased coronary artery disease and aor-
tic dissection [52-55], and increased risk of vascular calcifi-
cation in patients with type 2 diabetes mellitus [56].
VKORC1 genetic variations on chromosome 16 are not
in linkage disequilibrium with genetic variations known to
be associated with BMD through GWAS (Genome-Wide
Association Study) and candidate gene studies [28]. Moreo-
ver, many studies have investigated the effects of VKORC1
gene polymorphisms on bone. Spohn et al. [57] demon-
strated skeletal changes in a mouse VKORC1 knockout
model, supporting the idea that VKORC1 has an impact on
the skeleton. The bone phenotype of VKORC1-/- mice
showed a reduced length of the long bones. Mice lacking
VKORC1 in osteoblasts were viable and displayed a marked
reduction in Gla osteocalcin serum levels, and osteocalcin
failed to accumulate in the bone extracellular matrix in
VKORC1 osteoblast-specific knockout mice [18, 58].
Crawford et al. [28] suggested that common variants in
VKORC1 (rs9923231, rs9934438, rs2359612, rs8050894,
rs2884737, and rs7294) are indeed associated with BMD and
perhaps osteoporosis, but many of these results were limited
to African Americans. Fodor et al. [38] used a recessive
model and showed that there was a more frequent TT geno-
type of VKORC1 1173 C>T in patients with osteoporosis and
osteopenia. They found that the TT genotype occurred sig-
nificantly more frequently in the osteoporotic group than the
osteopenic group [38]. The frequency of 1173TT in Asian
patients was higher than in Caucasian and African popula-
tions [57, 59], and a VKORC1 polymorphism (TT genotype)
was associated with osteoporosis. Interethnic variability in
VKORC1 genotypes has been demonstrated. In our study, the
patient and control groups consisted of a Turkish population,
and all the patients and controls were Caucasians.
D’Andrea et al. [60] stated that the 1173TT genotype was
associated with less carboxylation of vitamin K-dependent
proteins. Osteocalcin and matrix Gla-protein, two members
of the Gla-protein family for which vitamin K acts as a co-
factor for gamma-glutamyl carboxylase, should exhibit a
lower level of carboxylation in this condition. The higher
prevalence of the TT genotype that was found in the osteo-
porotic group could be explained by this mechanism.
Holzer et al. [1] showed that the 3673 G>A genotype
influenced VKORC1 promoter activity and was associated
with the VKORC1 gene with BMD and fractures. They
showed that patients homozygous for 3673AA had slightly
lower BMD values than patients who carried AG or GG [1,
61]. In this study, we investigated the relationship between
the VKORC1 -1639G>A gene polymorphism and postmeno-
pausal osteoporosis. We showed that the GG genotype was
lower in the patients than the controls, but the difference was
not significant (P=0.135). The number of patients in our
study was smaller than in other genetic studies, which is a
limitation of our study. In addition to the number of patients
used, the osteoporotic index here presented are not showing
a clear/strong osteoporotic phenotype. It is clear if the T
score at the femur neck is considered (T=-2.76), but not if
considered the T scores at Femur Total or Lumbar. Other
limitations of this study are the lack of data on the genotype
distribution of VKORC1 -1639G>A in the Turkish popula-
tion, and already indicated is that the dietary VK intake of
patients is not known as well as the potential existence of
another polymorphisms in VK related genes (GGCX,
VKORC1L1 and PXR).
CONCLUSION
We showed that the VKORC1 -1639G>A polymorphism
might not be a risk factor for osteoporosis in postmenopausal
Table 2. Allele and genotype frequencies of the -1639 G>A polymorphisms in the VKORC1 gene
Patients (N=176) Control (N=140)
Genotype N Percent N Percent
2 P
Odds Ratio
(95% CI)
GG 45 25.57 47 33.57 2.42 0.135 0.68 (0.41-1.14)
GA 113 64.20 78 55.71 2.35 0.134 1.43 (0.88-2.30)
AA 18 10.23 15 10.71 0.020 1.000 0.95 (0.43-2.11)
Allele
G 203 57.67 172 61.43 0.913 0.86 (0.61-1.19)
A 149 42.33 108 38.57
0.370
2- Chi-square; N- Number of genotypes or alleles.
Vitamin K Epoxide Reductase -1639G>A Polymorphism Endocrine, Metabolic & Immune Disorders - Drug Targets, 2018, Vol. 18, No. 3 285
Turkish women. In addition to a larger population study,
more polymorphisms on VKORC1, nutritional VK status
and dietary intake of patients are further required to be as-
sessed to establish the association between the VKORC1 -
1639G>A polymorphism and postmenopausal osteoporosis.
ETHICS APPROVAL AND CONSENT TO
PARTICIPATE
This study was approved by the Local Ethics Committee
of Gaziosmanpasa University, Faculty of Medicine, Tokat,
Turkey (Reference Number: 13-KAK-066).
HUMAN AND ANIMAL RIGHTS
No animals were used in the study. The study on human
was carried out in accordance with the Declaration of Hel-
sinki (2013) of the World Medical Association.
CONSENT FOR PUBLICATION
A written informed consent was obtained from all subject
recruited.
CONFLICT OF INTEREST
The authors declare no conflict of interest, financial or
otherwise.
ACKNOWLEDGEMENTS
This work was supported by the Research Fund of
Gaziosmanpasa University (Project Number: 2013/18).
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